A Precision Therapy Approach for Retinitis Pigmentosa 11 Using Splice-Switching Antisense Oligonucleotides to Restore the Open Reading Frame of PRPF31.

Retinitis pigmentosa 11 is an untreatable, dominantly inherited retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 PRPF31. The expression level of PRPF31 is linked to incomplete penetrance in affected families; mutation carriers with higher PRPF31 expression can remain asymptomatic. The current study explores an antisense oligonucleotide exon skipping strategy to treat RP11 caused by truncating mutations within PRPF31 exon 12 since it does not appear to encode any domains essential for PRPF31 protein function. Cells derived from a patient carrying a PRPF31 1205C>A nonsense mutation were investigated; PRPF31 transcripts encoded by the 1205C>A allele were undetectable due to nonsense-mediated mRNA decay, resulting in a 46% reduction in PRPF31 mRNA, relative to healthy donor cells. Antisense oligonucleotide-induced skipping of exon 12 rescued the open reading frame with consequent 1.7-fold PRPF31 mRNA upregulation in the RP11 patient fibroblasts. The level of PRPF31 upregulation met the predicted therapeutic threshold of expression inferred in a non-penetrant carrier family member harbouring the same mutation. This study demonstrated increased PRPF31 expression and retention of the nuclear translocation capability for the induced PRPF31 isoform. Future studies should evaluate the function of the induced PRPF31 protein on pre-mRNA splicing in retinal cells to validate the therapeutic approach for amenable RP11-causing mutations.

1'-Acetoxychavicol Acetate from Alpinia galanga Represses Proliferation and Invasion, and Induces Apoptosis via HER2-signaling in Endocrine-Resistant Breast Cancer Cells.

Estrogen receptor-positive breast cancer patients have a good prognosis, but 30% of these patients will experience recurrence due to the development of resistance through various signaling pathways. This study aimed to evaluate the mode of anticancer effects of 1'-acetoxychavicol acetate, which is isolated from the rhizomes of Alpinia galanga in estrogen receptor positive (MCF7) human epidermal growth factor receptor 2-overexpressed (MCF7/HER2), and endocrine-resistant breast cancer cells (MCF7/LCC2 and MCF7/LCC9). 1'-Acetoxychavicol acetate showed antiproliferation in a concentration- and time-dependent fashion and had higher potency in human epidermal growth factor receptor 2-overexpressed cell lines. This was associated with down-regulation of human epidermal growth factor receptor 2, pERK1/2, pAKT, estrogen receptor coactivator, cyclin D1, and MYC proto-oncogene while in vivo and significant reduction in the tumor mass of 1'-acetoxychavicol acetate-treated zebrafish-engrafted breast cancer groups. The anti-invasive effects of 1'-acetoxychavicol acetate were confirmed in vitro by the matrigel invasion assay and with down-regulation of C - X-C chemokine receptor type 4, urokinase plasminogen activator, vascular endothelial growth factor, and basic fibroblast growth factor 2 genes. The down-regulation of urokinase plasminogen activator and fibroblast growth factor 2 proteins was also validated by molecular docking analysis. Moreover, 1'-acetoxychavicol acetate-treated cells exhibited lower expression levels of the anti-apoptotic Bcl-2 and Mcl-1 proteins in addition to enhanced stress-activated kinases/c-Jun N-terminal kinase 1/2 and poly-ADP ribose polymerase cleavage, indicating apoptotic cell induction by 1'-acetoxychavicol acetate. Moreover, 1'-acetoxychavicol acetate had higher potency in human epidermal growth factor receptor 2-overexpressed cell lines regarding its inhibition on human epidermal growth factor receptor 2, pAKT, pERK1/2, PSer118, and PSer167-ERα proteins. Our findings suggest 1'-acetoxychavicol acetate mediates its anti-cancer effects via human epidermal growth factor receptor 2 signaling pathway.

Antisense Oligonucleotide Induction of the hnRNPA1b Isoform Affects Pre-mRNA Splicing of SMN2 in SMA Type I Fibroblasts.

Spinal muscular atrophy (SMA) is a severe, debilitating neuromuscular condition characterised by loss of motor neurons and progressive muscle wasting. SMA is caused by a loss of expression of SMN1 that encodes the survival motor neuron (SMN) protein necessary for the survival of motor neurons. Restoration of SMN expression through increased inclusion of SMN2 exon 7 is known to ameliorate symptoms in SMA patients. As a consequence, regulation of pre-mRNA splicing of SMN2 could provide a potential molecular therapy for SMA. In this study, we explored if splice switching antisense oligonucleotides could redirect the splicing repressor hnRNPA1 to the hnRNPA1b isoform and restore SMN expression in fibroblasts from a type I SMA patient. Antisense oligonucleotides (AOs) were designed to promote exon 7b retention in the mature mRNA and induce the hnRNPA1b isoform. RT-PCR and western blot analysis were used to assess and monitor the efficiency of different AO combinations. A combination of AOs targeting multiple silencing motifs in hnRNPA1 pre-mRNA led to robust hnRNPA1b induction, which, in turn, significantly increased expression of full-length SMN (FL-SMN) protein. A combination of PMOs targeting the same motifs also strongly induced hnRNPA1b isoform, but surprisingly SMN2 exon 5 skipping was detected, and the PMO cocktail did not lead to a significant increase in expression of FL-SMN protein. We further performed RNA sequencing to assess the genome-wide effects of hnRNPA1b induction. Some 3244 genes were differentially expressed between the hnRNPA1b-induced and untreated SMA fibroblasts, which are functionally enriched in cell cycle and chromosome segregation processes. RT-PCR analysis demonstrated that expression of the master regulator of these enrichment pathways, MYBL2 and FOXM1B, were reduced in response to PMO treatment. These findings suggested that induction of hnRNPA1b can promote SMN protein expression, but not at sufficient levels to be clinically relevant.

Development of an engineered peptide antagonist against periostin to overcome doxorubicin resistance in breast cancer.

BACKGROUND: Chemoresistance is one of the main problems in treatment of cancer. Periostin (PN) is a stromal protein which is mostly secreted from cancer associated fibroblasts in the tumor microenvironment and can promote cancer progression including cell survival, metastasis, and chemoresistance. The main objective of this study was to develop an anti-PN peptide from the bacteriophage library to overcome PN effects in breast cancer (BCA) cells. METHODS: A twelve amino acids bacteriophage display library was used for biopanning against the PN active site. A selected clone was sequenced and analyzed for peptide primary structure. A peptide was synthesized and tested for the binding affinity to PN. PN effects including a proliferation, migration and a drug sensitivity test were performed using PN overexpression BCA cells or PN treatment and inhibited by an anti-PN peptide. An intracellular signaling mechanism of inhibition was studied by western blot analysis. Lastly, PN expressions in BCA patients were analyzed along with clinical data. RESULTS: The results showed that a candidate anti-PN peptide was synthesized and showed affinity binding to PN. PN could increase proliferation and migration of BCA cells and these effects could be inhibited by an anti-PN peptide. There was significant resistance to doxorubicin in PN-overexpressed BCA cells and this effect could be reversed by an anti-PN peptide in associations with phosphorylation of AKT and expression of survivin. In BCA patients, serum PN showed a correlation with tissue PN expression but there was no significant correlation with clinical data. CONCLUSIONS: This finding supports that anti-PN peptide is expected to be used in the development of peptide therapy to reduce PN-induced chemoresistance in BCA.

A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse.

Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by α-motor neuron loss in the spinal cord anterior horn. SMA results from deletion or mutation of the Survival Motor Neuron 1 gene (SMN1) and retention of SMN2. A single nucleotide difference between SMN1 and SMN2 results in exclusion of exon 7 from the majority of SMN2 transcripts, leading to decreased SMN protein levels and development of SMA. A series of splice enhancers and silencers regulate incorporation of SMN2 exon 7; these splice motifs can be blocked with antisense oligomers (ASOs) to alter SMN2 transcript splicing. We have evaluated a morpholino (MO) oligomer against ISS-N1 [HSMN2Ex7D(-10,-29)], and delivered this MO to postnatal day 0 (P0) SMA pups (Smn-/-, SMN2+/+, SMNΔ7+/+) by intracerebroventricular (ICV) injection. Survival was increased markedly from 15 days to >100 days. Delayed CNS MO injection has moderate efficacy, and delayed peripheral injection has mild survival advantage, suggesting that early CNS ASO administration is essential for SMA therapy consideration. ICV treatment increased full-length SMN2 transcript as well as SMN protein in neural tissue, but only minimally in peripheral tissue. Interval analysis shows a decrease in alternative splice modification over time. We suggest that CNS increases of SMN will have a major impact on SMA, and an early increase of the SMN level results in correction of motor phenotypes. Finally, the early introduction by intrathecal delivery of MO oligomers is a potential treatment for SMA patients.

Kluai Hin (Musa sapientum Linn.) peel as a source of functional polyphenols identified by HPLC-ESI-QTOF-MS and its potential antidiabetic function.

To date, information on the polyphenolic composition of Kluai Hin banana peel and pulp and the potential antidiabetic activity of its major active compounds is limited. This study aimed to identify polyphenols in extracts of fresh and freeze-dried Kluai Hin banana peel and pulp (methanol:water; M:W, 80:20 for flavonoids and acetone:water:acetic acid; A:W:A, 50:49:1 for phenolic acids) by RP-HPLC-DAD and HPLC-ESI-QTOF-MS. Additionally, inhibition of α-amylase and α-glucosidase activities was investigated with crude extracts from Kluai Hin banana peel and pulp, and compared with its major polyphenols ((+)-catechin, (-)-epicatechin and gallic acid) and the antidiabetic drug acarbose. (-)-Gallocatechin was the most abundant polyphenol and was detected in all fresh and freeze-dried pulp and peel extracts by RP-HPLC-DAD. Furthermore, unidentified polyphenol peaks of Kluai Hin were further explored by HPLC-ESI-QTOF-MS. The A:W:A fresh peel extract contained more total phenolic content (811.56 mg GAE/100 g) than the freeze-dried peel (565.03 mg GAE/100 g). A:W:A extraction of the fresh and freeze-dried peel of exhibited IC50 values for α-amylase activity 2.66 ± 0.07 mg/ml and 2.97 ± 0.00 mg/ml, respectively, but its inhibitory activity was lower than acarbose (IC50 = 0.25 ± 0.01 mg/ml). Peel extracts inhibited α-glucosidase activity, whereas pulp extracts had no effect. In addition, all standards, except gallocatechin, activated α-amylase activity, while, gallocatechin inhibited α-glucosidase activity better than acarbose. Therefore, we propose a further investigation into the use of Kluai Hin banana peel as a potential functional food for the management of postprandial glycaemic response to reduce diabetes risk and in the management of diabetes with a commercial drug.

Clinical expression and mitochondrial deoxyribonucleic acid study in twins with 14484 Leber's hereditary optic neuropathy: A case report.

BACKGROUND: This study aimed to explore clinical and molecular factors that cause discordance for clinical expression of Leber's hereditary optic neuropathy (LHON) in a pair of identical twins with the 14484 point mutation. CASE SUMMARY: Twin patients with the 14484 point mutation were studied for zygosity by using the Short Tandem Repeats Typing system. For the monozygotic twins, the radioactive restriction and densitometric analyses were used to quantitate the heteroplasmy level for the 14484 point mutation. The mitochondrial genome was analyzed to determine influential factors by mitochondrial deoxyribonucleic acid (DNA) sequencing, denaturing high-performance liquid chromatography and next generation sequencing. For the dizygotic twins, the nuclear DNA was analyzed. The twins with 14484 LHON were monozygotic with homoplasmy. No difference in the point mutation in mitochondrial DNA was found. No modifying genes that potentially influenced the disparity in phenotypic expression of LHON were detected in these twins. CONCLUSION: This 11-year follow-up of monozygotic twins showed additional genetic modifications and epigenetic factors are possibly associated with discordance for LHON.

Assessment of Biofilm Formation by Candida albicans Strains Isolated from Hemocultures and Their Role in Pathogenesis in the Zebrafish Model.

Candida albicans, an opportunistic pathogen, has the ability to form biofilms in the host or within medical devices in the body. Biofilms have been associated with disseminated/invasive disease with increased severity of infection by disrupting the host immune response and prolonging antifungal treatment. In this study, the in vivo virulence of three strains with different biofilm formation strengths, that is, non-, weak-, and strong biofilm formers, was evaluated using the zebrafish model. The survival assay and fungal tissue burden were measured. Biofilm-related gene expressions were also investigated. The survival of zebrafish, inoculated with strong biofilms forming C. albicans,, was significantly shorter than strains without biofilms forming C. albicans. However, there were no statistical differences in the burden of viable colonogenic cell number between the groups of the three strains tested. We observed that the stronger the biofilm formation, the higher up-regulation of biofilm-associated genes. The biofilm-forming strain (140 and 57), injected into zebrafish larvae, possessed a higher level of expression of genes associated with adhesion, attachment, filamentation, and cell proliferation, including eap1, als3, hwp1, bcr1, and mkc1 at 8 h. The results suggested that, despite the difference in genetic background, biofilm formation is an important virulence factor for the pathogenesis of C. albicans. However, the association between biofilm formation strength and in vivo virulence is controversial and needs to be further studied.

Immunopathogenesis of Emerging Candida auris and Candida haemulonii Strains.

The emergence of a multidrug-resistant Candida species, C. auris and C. haemulonii, has been reported worldwide. In Thailand, information on them is limited. We collected clinical isolates from Thai patients with invasive candidiasis. Both species were compared with a laboratory C. albicans strain. In vitro antifungal susceptibility and thermotolerance, and pathogenesis in the zebrafish model of infection were investigated. Both species demonstrated high minimal inhibitory concentrations to fluconazole and amphotericin B. Only C. auris tolerated high temperatures, like C. albicans. In a zebrafish swim-bladder-inoculation model, the C. auris-infected group had the highest mortality rate and infectivity, suggesting the highest virulence. The case fatality rates of C. auris, C. haemulonii, and C. albicans were 100%, 83.33%, and 51.52%, respectively. Further immunological studies revealed that both emerging Candida species stimulated genes involved in the proinflammatory cytokine group. Interestingly, the genes relating to leukocyte recruitment were downregulated only for C. auris infections. Almost all immune response genes to C. auris had a peak response at an early infection time, which contrasted with C. haemulonii. In conclusion, both emerging species were virulent in a zebrafish model of infection and could activate the inflammatory pathway. This study serves as a stepping stone for further pathogenesis studies of these important emerging species.

MTAP-related increased erythroblast proliferation as a mechanism of polycythaemia vera.

Polycythaemia vera (PV) is a haematological disorder caused by an overproduction of erythroid cells. To date, the molecular mechanisms involved in the disease pathogenesis are still ambiguous. This study aims to identify aberrantly expressed proteins in erythroblasts of PV patients by utilizing mass spectrometry-based proteomic analysis. Haematopoietic stem cells (HSCs) were isolated from newly-diagnosed PV patients, PV patients who have received cytoreductive therapy, and healthy subjects. In vitro erythroblast expansion confirmed that the isolated HSCs recapitulated the disease phenotype as the number of erythroblasts from newly-diagnosed PV patients was significantly higher than those from the other groups. Proteomic comparison revealed 17 proteins that were differentially expressed in the erythroblasts from the newly-diagnosed PV patients compared to those from healthy subjects, but which were restored to normal levels in the patients who had received cytoreductive therapy. One of these proteins was S-methyl-5'-thioadenosine phosphorylase (MTAP), which had reduced expression in PV patients' erythroblasts. Furthermore, MTAP knockdown in normal erythroblasts was shown to enhance their proliferative capacity. Together, this study identifies differentially expressed proteins in erythroblasts of healthy subjects and those of PV patients, indicating that an alteration of protein expression in erythroblasts may be crucial to the pathology of PV.

Targeted SMN Exon Skipping: A Useful Control to Assess In Vitro and In Vivo Splice-Switching Studies.

The literature surrounding the use of antisense oligonucleotides continues to grow, with new disease and mechanistic applications constantly evolving. Furthermore, the discovery and advancement of novel chemistries continues to improve antisense delivery, stability and effectiveness. For each new application, a rational sequence design is recommended for each oligomer, as is chemistry and delivery optimization. To confirm oligomer delivery and antisense activity, a positive control AO sequence with well characterized target-specific effects is recommended. Here, we describe splice-switching antisense oligomer sequences targeting the ubiquitously expressed human and mouse SMN and Smn genes for use as control AOs for this purpose. We report two AO sequences that induce targeted skipping of SMN1/SMN2 exon 7 and two sequences targeting the Smn gene, that induce skipping of exon 5 and exon 7. These antisense sequences proved effective in inducing alternative splicing in both in vitro and in vivo models and are therefore broadly applicable as controls. Not surprisingly, we discovered a number of differences in efficiency of exon removal between the two species, further highlighting the differences in splice regulation between species.

Rational design of antisense oligomers to induce dystrophin exon skipping.

Duchenne muscular dystrophy (DMD), one of the most severe neuromuscular disorders of childhood, is caused by the absence of a functional dystrophin. Antisense oligomer (AO) induced exon skipping is being investigated to restore functional dystrophin expression in models of muscular dystrophy and DMD patients. One of the major challenges will be in the development of clinically relevant oligomers and exon skipping strategies to address many different mutations. Various models, including cell-free extracts, cells transfected with artificial constructs, or mice with a human transgene, have been proposed as tools to facilitate oligomer design. Despite strong sequence homology between the human and mouse dystrophin genes, directing an oligomer to the same motifs in both species does not always induce comparable exon skipping. We report substantially different levels of exon skipping induced in normal and dystrophic human myogenic cell lines and propose that animal models or artificial assay systems useful in initial studies may be of limited relevance in designing the most efficient compounds to induce targeted skipping of human dystrophin exons for therapeutic outcomes.

Cryptococcus neoformans/gattii Species Complexes from Pre-HIV Pandemic Era Contain Unusually High Rate of Non-Wild-Type Isolates for Amphotericin B.

INTRODUCTION: The Cryptococcus neoformans/gattii species complexes are a leading cause of fatality among HIV-infected patients. Despite the unavailability of clinical breakpoints (CBPs) for antifungal agents, epidemiological cutoff values (ECVs) were recently proposed, and non-wild-type isolates for polyenes and azoles are being increasingly reported. However, the distributions of the susceptibility patterns for pre-HIV-era isolates have not been studied. METHODS: We determined the in vitro antifungal susceptibility patterns of 233 Cryptococcus isolates, collected at the National Institutes of Health, USA, in pre-HIV pandemic era, to study minimum inhibitory concentrations (MICs) to the important drugs for cryptococcosis and to compare the results with strain genotypes. Amphotericin B susceptibility was compared to published ECV of C. neoformans. RESULTS: The 233 Cryptococcus strains consisted of 89.7% C. neoformans species complex and 10.3% C. gattii species complex. Most were from clinical sources (189, 81.1%), and the major molecular type was VNI (146, 62.7%). The highest geometric mean (GM) was observed for fluconazole (GM = 0.96 µg/mL) while the lowest was for itraconazole (GM = 0.10 µg/mL). MICs to fluconazole in C. gattii species complex were significantly higher than C. neoformans species complex (p < 0.001). Moreover, C. neoformans/VNI strains showed significantly higher MICs than others such as C. neoformans/VNII to fluconazole (p < 0.0001) and C. deneoformans/VNIV to amphotericin B (p = 0.022) and fluconazole (p = 0.008). In our collection of 167 clinical C. neoformans species complex strains, 85 (50.9%), 24 (14.4%), and 3 (1.8%) strains had an amphotericin B (AMB)-MIC of 1, 2, and 4 µg/mL, respectively. The high percentage (66.9%, 79/118 strains) of non-wild-type clinical C. neoformans VNI strains, using an AMB-ECV of 0.5 µg/mL, was found. Moreover, 25 of 28 (89.3%) C. neoformans VNI strains from environmental and veterinary sources also had AMB-MICs above 0.5 µg/mL. In general, there was no significant difference in GM AMB-MIC of the clinical strains isolated from patients with (35 patients) and without (78 patients) prior AMB treatment (0.85 vs 0.76; p = 0.624). GM MIC of the environmental strains was not significantly different from that of the prior AMB-treatment strains (0.98 vs 0.76, p = 0.159) and the post-AMB-treatment strains (0.98 vs 0.85, p = 0.488). CONCLUSION: The high rate of non-wild-type among these otherwise naive isolates to amphotericin B is unexpected. Confirmation with more strains from a later era is needed.

Generation of three induced pluripotent stem cell lines from a patient with Usher syndrome caused by biallelic c.949C > A and c.1256G > T mutations in the USH2A gene.

Mutations in the USH2A gene are the most common cause of Usher syndrome and autosomal recessive non-syndromic retinitis pigmentosa. Here, we describe the generation of three induced pluripotent stem cell lines from dermal fibroblasts derived from a patient carrying biallelic c.949C > A and c.1256G > T variants in the USH2A gene, using episomal reprogramming plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28, mir302/367 and shRNA targeting TP53. All three lines expressed pluripotency markers, displayed unaltered karyotypes as well as trilineage differentiation potential, and were negative for reprogramming episomes and mycoplasma.

By-passing the nonsense mutation in the 4 CV mouse model of muscular dystrophy by induced exon skipping.

BACKGROUND: Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein-truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg-Dmd(mdx-4Cv)/J (4(CV)) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame. METHODS: A series of 2'-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels. RESULTS: Compared to the original mdx mouse studies, induction of exon skipping from the 4(CV) dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near-full length dystrophin protein in cultured 4(CV) myogenic cells and in vivo, after a single intramuscular injection. CONCLUSIONS: By-passing the protein-truncating mutation in the 4(CV) mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo.

Consequences of Making the Inactive Active Through Changes in Antisense Oligonucleotide Chemistries.

Antisense oligonucleotides are short, single-stranded nucleic acid analogues that can interfere with pre-messenger RNA (pre-mRNA) processing and induce excision of a targeted exon from the mature transcript. When developing a panel of antisense oligonucleotides to skip every dystrophin exon, we found great variation in splice switching efficiencies, with some antisense oligonucleotides ineffective, even when directed to canonical splice sites and transfected into cells at high concentrations. In this study, we re-evaluated some of these ineffective antisense oligonucleotide sequences after incorporation of locked nucleic acid residues to increase annealing potential. Antisense oligonucleotides targeting exons 16, 23, and 51 of human DMD transcripts were synthesized as two different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone or mixmers containing several locked nucleic acid residues, which were then transfected into primary human myotubes, and DMD transcripts were analyzed for exon skipping. The ineffective 2'-O-methyl modified antisense oligonucleotides induced no detectable exon skipping, while all corresponding mixmers did induce excision of the targeted exons. Interestingly, the mixmer targeting exon 51 induced two unexpected transcripts arising from partial skipping of exon 51 with retention of 95 or 188 bases from the 5' region of exon 51. These results indicated that locked nucleic acid/2'-O-methyl mixmers are more effective at inducing exon skipping, however, this improvement may come at the cost of activating alternative cryptic splice sites and off-target effects on gene expression.

Antisense Oligonucleotide-Mediated Terminal Intron Retention of the SMN2 Transcript.

The severe childhood disease spinal muscular atrophy (SMA) arises from the homozygous loss of the survival motor neuron 1 gene (SMN1). A homologous gene potentially encoding an identical protein, SMN2 can partially compensate for the loss of SMN1; however, the exclusion of a critical exon in the coding region during mRNA maturation results in insufficient levels of functional protein. The rate of transcription is known to influence the alternative splicing of gene transcripts, with a fast transcription rate correlating to an increase in alternative splicing. Conversely, a slower transcription rate is more likely to result in the inclusion of all exons in the transcript. Targeting SMN2 with antisense oligonucleotides to influence the processing of terminal exon 8 could be a way to slow transcription and induce the inclusion of exon 7. Interestingly, following oligomer treatment of SMA patient fibroblasts, we observed the inclusion of exon 7, as well as intron 7, in the transcript. Because the normal termination codon is located in exon 7, this exon/intron 7-SMN2 transcript should encode the normal protein and only carry a longer 3' UTR. Further studies showed the extra 3' UTR length contained a number of regulatory motifs that modify transcript and protein regulation, leading to translational repression of SMN. Although unlikely to provide therapeutic benefit for SMA patients, this novel technique for gene regulation could provide another avenue for the repression of undesirable gene expression in a variety of other diseases.

Antisense oligonucleotide induction of progerin in human myogenic cells.

We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate the role of progerin in premature muscle ageing.

A novel morpholino oligomer targeting ISS-N1 improves rescue of severe spinal muscular atrophy transgenic mice.

In the search for the most efficacious antisense oligonucleotides (AOs) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (-10, -34), PMO18 (-10, -27), and PMO20 (-10, -29), complementary to the SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon 7 inclusion in vitro in spinal muscular atrophy (SMA) patient fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further in a mouse model of severe SMA. After a single intracerebroventricular (ICV) injection in neonatal mice, PMO25 increased the life span of severe SMA mice up to 30-fold, with average survival greater by 3-fold compared with PMO18 at a dose of 20 µg/g and 2-fold at 40 µg/g. Exon 7 inclusion was increased in the CNS but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon 7 inclusion both in the CNS and peripherally. Systemic administration of a 10-µg/g concentration of PMO25 conjugated to an octaguanidine dendrimer (VMO25) increased the life span only 2-fold in neonatal type I SMA mice, although it prevented tail necrosis in mild SMA mice. Higher doses and ICV injection of VMO25 were associated with toxicity. We conclude that (1) the 25-mer AO is more efficient than the 18-mer and 20-mer in modifying SMN2 splicing in vitro; (2) it is more efficient in prolonging survival in SMA mice; and (3) naked Morpholino oligomers are more efficient and safer than the Vivo-Morpholino and have potential for future SMA clinical applications.

Improved antisense oligonucleotide design to suppress aberrant SMN2 gene transcript processing: towards a treatment for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, resulting in reduced SMN protein. Humans possess the additional SMN2 gene (or genes) that does produce low level of full length SMN, but cannot adequately compensate for loss of SMN1 due to aberrant splicing. The majority of SMN2 gene transcripts lack exon 7 and the resultant SMNΔ7 mRNA is translated into an unstable and non-functional protein. Splice intervention therapies to promote exon 7 retention and increase amounts of full-length SMN2 transcript offer great potential as a treatment for SMA patients. Several splice silencing motifs in SMN2 have been identified as potential targets for antisense oligonucleotide mediated splice modification. A strong splice silencer is located downstream of exon 7 in SMN2 intron 7. Antisense oligonucleotides targeting this motif promoted SMN2 exon 7 retention in the mature SMN2 transcripts, with increased SMN expression detected in SMA fibroblasts. We report here systematic optimisation of phosphorodiamidate morpholino oligonucleotides (PMO) that promote exon 7 retention to levels that rescued the phenotype in a severe mouse model of SMA after intracerebroventricular delivery. Furthermore, the PMO gives the longest survival reported to date after a single dosing by ICV.